Electron microscope

ABSTRACT

The present invention provides an analysis of displacement by calculating the phase variance image P′ (k, l) between Fourier transformed images of paired images S1 (n, m) and S2 (n, m) to determine the center of gravity of δ peak appearing on the invert Fourier transform image of the images. The present invention provides numerous advantages such as a precision of displacement analysis of a fraction of pixel to thereby allow to improve the precision of focal analysis, or reduced number of pixels required to achieve the same precision, evaluation of reliability of the analysis by using the δ peak intensity, influence of varying background reduced by using a phase variance component. The improved performance by the present invention allows any operator skilled or not to achieve a best focusing.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention:

[0002] The present invention relates to an apparatus for automaticallycompensating for the focus and the amount of displacement using anelectron microscope image.

[0003] 2. Description of the Prior Art:

[0004] The inventor of the present invention have searched the Prior Artwith respect to apparatuses for compensating for the focus and theamount of displacement by determining whether or not to automaticallycompensate therefor by using an electron microscope image, or methodsfor compensating for the amount of displacement of a continuously movingspecimen stage. As the result of search, there have been found threerelevant topics. First, a paper entitled as “The correction of imagedrift for autotuning a TEM using phase spectrum” by Norihiko Ichise etal., disclosed in the proceedings of the 51st academic conference ofJapan Electron Microscope Association, May 1995, pp. 161, discloses amethod for analyzing and compensating for the influence of image driftby using the phase spectrum method for analyzing the focus, astigmatism,and shifted axis. However, this paper does disclose nothing about theimprovement of the precision of analysis by means of the computation ofthe gravity center of a peak, and determination by means of compensationvalues, as well as about the compensation of the focus and drift of acontinuously moving specimen stage. Second, the Japanese UnexaminedPatent Publication No. Hei 10-187993, discloses an apparatus foranalysis of displacement between images by using the phase variance ofFourier transform images on two photos taken in different conditions.

[0005] However, this application does disclose nothing about thefeedback to an electron microscopy apparatus and the spirit of the artbut only the measurement of shape and distance of an object from a markattached to the object. Third, the Japanese Unexamined PatentPublication No. Hei 10-339607, discloses the detection of amount ofdisplacement by image processing of the parallax of electron microscopyimages, and the feedback of the result thereof to the electronmicroscopy apparatus. More specifically, the images do not move beforeand after the angle of incidence of electron beam varies if a specimenis located just on the focus plane, however, if the specimen is locatedout of focusing plane then the images move before and after the angle ofincidence of electron beam. The relationship between the displacement Dand out-of-focus F is D=M α (F+Cs α²), where α designates to thedeflection angle of incident electron beam, M to the magnification rate,Cs to the coefficient of spherical aberration, therefore theout-of-focus F may be given if the parallax displacement D isdetermined. There is disclosed an apparatus for compensating for thefocusing of objective lens system by storing on a memory a pair ofimages before and after altering the incident angle to apply across-correlation method or least-squares estimation method to analyzethe displacement D to determine the amount of defocus F. However, theanalysis method of displacement using the phase variance of Fouriertransform images is not disclosed. In an apparatus for automaticallycompensating for the focusing or the amount of displacement by usingelectron microscopy images, the performance may depend on the settingsof the photographic condition of images, analyzing images, and feedingback the analysis results. However, the optimization is not performedwith respect to the object, precision and time of compensation.

[0006] The performance of the apparatus for automatic focusing anelectron microscope in accordance with the displacement D betweenelectron microscopy images such as the focus analysis using parallax andthe like may depend largely on the analysis method of displacement D.The analysis methods of displacement used heretofore, such as forexample the cross-correlation method, least-squares method and the like,were limited in terms of the precision by size of pixels of the electronbeam detector. The length of a side of pixel of a CCD camera used forpresent electron microscopy imaging is approximately 25 microns. Theamount of defocus F corresponding to a pixel may depend on the angle andmagnitude of incident electron beam, the variance of incident angle αmay be approximately at most 0.5° due to the limitation by the holediameter of objective aperture, and the magnitude should be the actualobservation magnitude. For example, at a magnification of 5,000, and anincident angle variance of 0.5°, the focusing distance corresponding tothe displacement D of a pixel is approximately 0.6 microns. This valueis less than the precision level of focus compensation by a skilledoperator. The improvement of performance of apparatus such as refiningthe images used by the displacement analysis in favor of improving theprecision in the focusing analysis may cause excessive time spent foranalysis and excessive cost of hardware, thus is not practical.

[0007] The conventional method of displacement analysis has nofunctionality of numerical verification on whether or not an analysishas been performed correctly, so that the operator had to guess theresult by the eye. Otherwise the operator had to compensate for thefocusing based on thus obtained analysis results to verify that thecompensation was done accurately. Since the automatic compensator hasnot assurance enough to correctly perform any analysis, there may be aneed for a functionality of aborting compensation when the result ofanalysis is not highly reliable.

[0008] Furthermore, in the analysis of displacement in the prior art theanalysis was almost impossible when the image was shadowed by theobjective aperture. This phenomenon may be encountered routinely in theTEM observation, malfunction thereby thus may cause problems inpractice.

SUMMARY OF THE INVENTION

[0009] An object of the present invention is to provide an apparatus forautomatic compensation by determining whether or not the focusing or theamount of movement is to be automatically compensated for by usingelectron microscope images.

[0010] The present invention uses the analysis method for determiningthe displacement of electron microscope images as will be describedbelow.

[0011] For a pair of images containing displacement, a deflector meansfor deflecting the incident angle to first electron microscope specimenis used to obtain a pair of images, to perform a Fourier transformthereon to compute the phase variant images. The analysis imagesobtained by having an invert Fourier transform or Fourier transformapplied on the phase variant images may contain δ peaks at the positionscorresponding to the displacement. The analysis images may be assumed tocontain only the δ peaks, so that any peaks other than δ may be treatedas noise component.

[0012] Therefore, the computation of the gravity center of δ peaks willresult in an accurate position of the peaks even when the positions of δpeaks contains fractional component. The intensity of δ peaks computedafter normalizing the intensity of analysis images may be used as acorrelation, which indicates a match between images.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The accompanying drawings, which are incorporated in andconstitute a part of this specification illustrate embodiments of theinvention and, together with the description, serve to explain theobjects, advantages and principles of the invention. In the drawings,

[0014]FIG. 1 shows a schematic block diagram of an automatic focusingmethod using parallax;

[0015]FIGS. 2A and 2B show schematic diagrams of screen display examplesfor setting parameters, and displaying the analysis result of thefocusing;

[0016]FIG. 3 shows a flow chart of TEM imaging processes;

[0017]FIG. 4 shows a schematic diagram illustrating the principal offocusing method using parallax;

[0018]FIG. 5 shows a schematic diagram illustrating the computation ofdisplacement;

[0019]FIG. 6 shows a schematic diagram of basic configuration of anelectron detector 17 for TEM;

[0020]FIGS. 7A to 7C show schematic diagrams illustrating the positionand intensity of peaks in analysis images, in case of a high contrastspecimen, a low contrast specimen, and a specimen with smalldisplacement, respectively;

[0021]FIGS. 8A and 8B show flow charts illustrating the identificationof peaks corresponding to the displacement from analysis images, in caseof a process using a mask at the origin and a process outputting twopeaks, respectively;

[0022]FIG. 9 shows a flow chart illustrating the focusing;

[0023]FIG. 10 shows a flow chart illustrating the automatic analyzerusing a TEM;

[0024]FIGS. 11A to 11D, respectively, show schematic diagramsillustrating the identification of analyzing areas by determining thedirection and the shape of a mesh 22, in case of a TEM image of the mesh22 at a lower magnification power, an image labeled and binary coded,the relationship between the gravity center 24 of holes 23 and thedirection of mesh 22, and identified holes 23 each of which is splitinto a plurality of analysis areas;

[0025]FIG. 12 shows a flow chart illustrating analysis of the directionand shape of a mesh;

[0026]FIGS. 13A and 13B show schematic diagrams illustrating determiningthe presence and absence of specimen in a TEM image, in each case of aTEM image and image intensity histogram thereof when a specimen ispresent, and when no specimen is present;

[0027]FIG. 14 shows a flow chart illustrating viral inspection in ananalysis area;

[0028]FIG. 15 shows a display screen used for setting the parameters anddisplaying the progress of analysis in an automatic analysis;

[0029]FIGS. 16A to 16D show display screen examples for displaying theresults of automatic analysis;

[0030]FIGS. 17A and 17B show schematic diagrams illustrating determiningthe presence and absence of specimen in a TEM image, in each case of aTEM image and the distribution of high frequency component of theFourier transform image thereof when a specimen is present, and when nospecimen is present;

[0031]FIGS. 18A to 18C show schematic diagrams illustrating the positionof observing field, positional setting of image shift, positionalsetting of specimen stage in the course of elapsed time, when theanalysis area is shifted by moving a specimen on the specimen stage 18,image shifting by means of deflective coil 16 for condenser system, andimage shifting by means of both the movement of specimen and imageshifting, respectively;

[0032]FIG. 19 shows an electron microscope in accordance with thepresent invention;

[0033]FIG. 20 shows a process of viral analysis using the TEM;

[0034]FIG. 21 shows a process of evaluation of semiconductors using theTEM;

[0035]FIG. 22 shows a process of trimming from an TEM image patternssubjected to be inspected and of comparing one with another thereof;

[0036]FIG. 23 shows a sequence of analysis when shifting inspectionfield by means of the specimen stage 18 or deflector coil 16 forcondenser system;

[0037]FIG. 24 shows a schematic diagram illustrating the principal ofthe method of focus analysis using the parallax, (a) when irradiating aspecimen with an electron beam in parallel at the focal position, (b)when irradiating diagonally a specimen with an electron beam at thefocal position, (c) when irradiating diagonally an electron beam to aspecimen at the position out of focus;

[0038]FIG. 25 shows a schematic diagram illustrating a process forautomatic focusing using the parallax;

[0039]FIG. 26 shows a schematic diagram illustrating a process forautomatic focusing the displacement of a specimen by the deflector coil16 for condenser system;

[0040]FIG. 27 shows a schematic diagram illustrating a process forimproving the precision of displacement analysis of a specimen by usinga zoom lens system;

[0041]FIG. 28 shows a schematic diagram illustrating the focaldistribution around the optical axis in an electron beam astigmaticlens; and

[0042]FIG. 29 shows a schematic diagram illustrating a process forcompensating for astigmatism in an electron microscope.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0043] A detailed description of preferred embodiments embodying thepresent invention will now be given referring to the accompanyingdrawings.

[0044] [First Embodiment]

[0045]FIG. 19 shows an transmission electron microscope (referred to andabbreviated to as TEM hereinafter) used in the embodiments disclosedherein in accordance with the present invention.

[0046] The TEM comprises an electron gun 11 and electron gun controlcircuit 11′, a condenser lens 12 and condenser lens control circuit 12′,a deflective coil for condenser system 13 and deflective coil controlcircuit for condenser system 13′, an objective lens 14 and objectivelens control circuit 14′, a projector lens 15 and projector lens controlcircuit 15′, a deflective coil for condenser system 16 and deflectivecoil control circuit for condenser system 16′, an electron detector 17and electron detector control circuit 17′, a specimen stage 18 andspecimen stage control circuit 18′, a computer with control software andimage processing software 19. Each of control circuits may receivecontrol commands sent from the control software in the computer 19,perform controls and return the return value to the computer. Theelectron detector 17 is a detector constituted of a plurality of pixelssuch as a CCD camera, which may transmit signals of obtained imagesthrough the cable for image transmission to the storage device of thecomputer 19 or to the displacement analysis processor using phasevariance of Fourier transform images 20. The displacement analysisprocessor using phase variance of Fourier transform images 20 isconnected to the computer with control software and image processingsoftware 19.

[0047]FIG. 3 shows a flow chart of TEM imaging. An acceleration voltageis applied to the electron beam that is the first charged particle beamgenerated by the electron gun 11, then the deflective coil for condensersystem 13 as a deflector means is used for adjusting the deflection ofbeam such that the electron beam passes through the optical axis, toverify that the electron beam reaches to the electron detector 17. Inthis document ‘z’ axis is defined as an axis in parallel to the opticalaxis, x-y plane is defined as the plane normal to the optical axis.After adjusting the condenser lens 12, a specimen 21 is set into theTEM, and a TEM image at lower magnification rate is observed. Theobjective aperture is inserted to the optical axis in order to increasethe contrast of TEM image. By gradually increasing the magnification ofthe projector lens 15, an observation field is selected and the focusingis adjusted to take an image of a second charged particle beam that isan electron beam transmitted through the specimen by the electrondetector 17.

[0048] To the analysis of focusing in the above focusing step is applieda focusing analysis method using parallax. In this method a first TEMimage obtained by an electron beam emitted at first incident angle inalmost parallel to the optical axis, and a second TEM image obtained byan electron beam emitted at second incident angle descend to an angle αfrom the optical axis are used. As shown in FIG. 4, when the beam isfocused somewhere out of focus, there will be generated somedisplacement between the first TEM image and second TEM image. Thedefocus F is correlated with displacement D by parallax in therelationship given by D=M α(F+Cs α²) The magnification rate M and theangle α may be set by the operator. Since the coefficient of sphericalaberration Cs is intrinsic to a specific apparatus, the amount ofdefocusing F may be specified by determining the displacement D of theimage pair. The present invention is characterized in that an analysismethod based on the phase variant analysis of Fourier transform imagesis applied to the determination of displacement D. As shown in FIG. 1,first and second TEM images may be obtained by using the electrondetector 17, with the angle of incidence of electron beam with respectto the specimen being varied by using the deflective coil for condensersystem 13 mounted above the objective lens 14. The first and second TEMimages thus obtained will be sent to the displacement analysis processorusing phase variance of Fourier transform images 20, from which thedisplacement D will be further sent to the computer with controlsoftware and image processing software 19. The computer 19 will computethe amount of defocus F from the displacement D to determine the currentof objective lens I_(obj) required for adjusting the focusing, andfinally the focus of the objective lens 14 will be thereby compensatedfor.

[0049]FIG. 5 shows a schematic diagram illustrating the computation ofdisplacement using the phase component of Fourier transform. For a pairof images with a displacement D=(dx, dy), by assuming S1 (n, m)=S2(n+dx, m+dy) the two dimensional discrete Fourier transform of S1 (n, m)and S2 (n, m) will be S1′ (k, l), and S2′ (k, l).

[0050] In accordance with the formula F {S (n+dx, m+dy))=F {S (n, m)}exp (idxk+idyl) of the Fourier transform, S1 (k, l)=S2′ (k, l) exp(idxk+idyl) maybe obtained. The displacement in S1′ (k, l) and S2′ (k,l) above may be expressed as the phase variance exp (idxk+idyl)=P′ (k,l). Since P′ (k, l) is a wave with the cycle (dx, dy), then in an imageP (n, m) which is subjected to invert Fourier transform of a phasevariant image P′ (k, l), a δ peak will be appeared at the location (dx,dy). If (dx, dy) have a fraction, for example (dx, dy)=(2.5, 2.5), thenthe intensity of δ peak will be distributed in a manner aliquot to (2,2), (2, 3), (3, 2), and (3, 3). Since it can be assumed that in theimage P (n, m) only the δ peak may be present, the computation of fourgravity centers of intensity of pixels allows the correct determinationof δ peak even if a fraction is present. The cross-correlation method,which is used in the Prior Art as an analysis method, uses |S1′| and|S2′| as analysis images to compute the displacement based on thelocation with the maximum value in the images. Since the analysis imagescontains, together with information with respect to the displacement,any image intensity, i.e., the information about amplitude, theprecision of analysis will not be improved by the computation of thegravity center. It should be noted here that, when the information onamplitude is not totally eliminated, if an image is computed with theamplitude component suppressed by performing log or {square root}{squareroot over ( )} on the amplitude component of S1′ (k, l)*S2′ (k,l)*=|S1′| |S2′| exp (idxk+idyl) and then with the invert Fouriertransform applied, a δ peak will be appeared at the position (dx, dy) ofdisplacement vector, the analysis of displacement may be performed basedon the image. Also it should be noted that a δ peak will be appeared atthe position (−dx, −dy) if the phase variant image P′ (k, l) is Fouriertransformed, so that the analysis of displacement may be performed onthe Fourier transformed image of the phase variant image P′ (k, l).Furthermore, any one of other orthogonal transformations may be usedinstead of Fourier transform to compute an image with peak correspondingto the displacement.

[0051] The analysis of displacement may be allowed if common componentis present sufficiently in S1 (n, m) and S2 (n, m) when the variance inS1 (n, m) and S2 (n, m) includes not only the displacement but also thevariable noise component or background behaviors, or when the image isdeformed more or less due to the change of angle of incidence ofelectron beam. In such a case any peaks other than δ peak may be treatedas noises. When the peak intensity δ is computed after normalizing theintensity of the entire image P (n, m), the intensity will be weaken ifthe unmatched area in the pair of images is larger, in other words, ifthe noise increases. Since the peak intensity will be stronger if theimages in the pair match in a larger extent, whereas the intensity willbe weaker if the images match in a smaller extent, the operator mayidentify the signal noise ratio, namely the reliability of results ofanalysis by expressing the peak intensity as the correlation valueindicating the match between images in the pair. In addition,malfunction may be prevented by setting the lower threshold value of thecorrelation to cause the adjustment of objective lens not to beperformed in case in which computed correlation value is less than thelower threshold value.

[0052] The analysis of displacement as described above has further anadvantage that it is hardly affected by the variance in the backgroundsince it uses the phase component of images. In the Prior Art, imageanalysis may not be allowed if there is any variance in the backgrounddue to for example the distribution of intensity of irradiation current,while the analysis of displacement in accordance with the presentinvention may be allowed in the same condition. Also, the image analysismay not be allowed in the conventional analysis methods if the imagecontains for example the shadow of objective aperture, the analysis ofdisplacement in accordance with the present invention may be allowed ifthe common area of the pair of images is sufficiently presented evenwhen the shadow of objective aperture is contained in some extent. As itis anticipated that a user not skilled in the TEM operation may use theautomatic focusing apparatus, it may be important that the TEMauto-focus works even when the fine adjustment of TEM is somewhatincomplete.

[0053] In order to perform the analysis of displacement, TEM images maybe captured by the electron detector 17 such as a CCD camera. The signaldetected by the electron detector 17 is amplified by an amplifier, thenquantized to send to the computer 19 or the displacement analysisprocessor using phase variance of Fourier transform images 20. It shouldbe noted that it is important to appropriately set the gain and offsetof the amplifier, otherwise almost all characteristics contained in theimages will be eliminated in the course of quantization. The electrondetector 17 comprises a functionality of automatic adjustment of gainand offset of the detector amplifier by computing the mean intensityvalue and dispersion of images so as to settle to specified values.Because it is anticipated that the specified mean and dispersion may notbe obtained by the gain and offset used, the detector comprises anotherfunctionality which may warn the operator when the contrast adjustmentis not complete to ask for further adjustments such as redefining theviewing field or tuning of TEM itself.

[0054]FIG. 6 shows a schematic diagram of basic configuration of anelectron detector 17 for TEM. The electron detector 17 comprises ascintillator 71, a photo coupler 72, and a CCD camera 73. The electronsemitted to the scintillator 71 generate photons. Thus generated photonswill pass through the photo coupler 72, which is made of a plurality ofbundled optical fibers, to the CCD camera 73 with positional informationbeing held. The CCD camera 73 is constituted of a two dimensional arrayof a plurality of pixels. The charge generated by the photons incomingto the CCD camera 73 will be stored in each pixel. The stored chargewill be read out as the output signal from each pixel. The gain of eachpixel, in other words the intensity of signal output delivered by oneincident electron may be determined by the light emitting efficiency ofthe scintillator 71, transmission efficiency of the photo coupler 72,and the quantization efficiency of the CCD camera 73. Because theseconstants are not uniform from one pixel to another, a fixed pattern ispreformed in the electron detector 17.

[0055] The image captured by the electron detector 17 with a fixedpattern may store a first contrast corresponding to the specimenstructure together with a second contrast corresponding to the fixedpattern of the electron detector 17. When applying the analysis ofdisplacement to the image captured by the electron detector 17 with afixed pattern, the first contrast corresponding to the structure ofspecimen may move between paired images S1 (n, m) and S2 (n, m), whileon the other hand the second contrast corresponding to the fixed patternof the electron detector 17 does not, so that in the analysis image P(n, m) a first peak caused by the specimen structure will be observed atthe position relative to the displacement, while a second peak caused bythe fixed pattern will be observed at the origin. A very low contrastimage of specimen structure such as a TEM image may often have theintensity of second peak caused by the fixed pattern larger than theintensity of first peak caused by the specimen structure. The images towhich the displacement analysis has been applied heretofore were highcontrast, high sharpness images obtained by an optical apparatus, withthe effect of fixed pattern almost neglected such that the maximumintensity peak in the analysis image P (n, m) was determined to be theresult of analysis. However, as the TEM images have considerable effectof the fixed pattern, the second peak caused by the fixed pattern mightbe determined as the result of analysis if the conventional peakdetection method is applied in which the maximum intensity peak is theresult of analysis, such that the analysis may often be complete withoutdisplacement detected.

[0056] A preprocess, such as normalizing of gains, for minimizing theeffect of fixed pattern, by subtracting images by a fixed patternpreviously captured, may be incorporated in the CCD camera controllingsoftware. The fixed pattern used for the computation should be updatedat a predetermined interval because the pattern is affected by aging. Inorder to minimize the influence of the fixed pattern the routinemaintenance is indispensable. The change of fixed pattern due to thedifference in capturing conditions such as the amount of irradiation ofelectron beam and the like is inevitable if the apparatus is routinelymaintained. Although using only the normalization of gain may reduce theinfluence of fixed pattern, it will be difficult to completely eliminateit. Images of low contrast specimen structure such as electronmicroscopy images may have the second peak intensity larger than thefirst peak intensity of specimen structure even if any image processingsuch as gain normalization is performed on the paired images S1 (n, m)and S2 (n, m).

[0057] It is necessary for the displacement analysis of TEM images toadd a step of automatically determining the first peak caused by thespecimen structure from the analysis image having the first peak causedby the specimen structure together with the second peak caused by thefixed pattern. There are two algorithms as described below for automaticpeak detection. Both algorithms make use of the fact that the secondpeak caused by the fixed pattern is observed at the origin.

[0058] Since the second peak intensity caused by the fixed patternappears at the origin, first algorithm applies a masking of the peak atthe origin for substituting the intensity at the origin with a zero or apredetermined value. However, since the first peak may appear at theorigin, the application of the exception at the origin is required to bedetermined. Now referring to a specific example shown in FIG. 7, theflow of peak detection will be described below. It is now assumed theposition D1 and intensity I1 of the first peak 31 caused by the specimenstructure, and the position D2 and intensity I2 of the second peak 32caused by the fixed pattern. Since D2=0, there are predicted cases of|D1|>0 AND I1>I2 (FIG. 7 A) , |D1|>0 AND I1<I2 (FIG. 7B), and |D1˜=0(FIG. 7C). For each case the intensity I of the maximum intensity peak33 retrieved by normalizing the analysis image P (n, m) will be comparedwith the intensity I′ of the maximum intensity peak 34 retrieved bynormalization after exception of (masking of) the peak in the analysisimage P (n, m) at the origin. In case of FIG. 7A, if the intensity ofsecond peak is as small as it can be neglected, the intensity of themaximum intensity peak will be the same before and after exception ofthe peak at the origin, whereas if the intensity of second peak isstrong enough then the intensity allocated to the second peak 32 byexcepting the peak at the origin moves to the first peak 31 and noise35, resulting in I≦I′. In case of FIG. 7B, without exception of the peakat the origin, the intensity allocated to the first peak 31 and thesecond peak 32 will be consolidated to the first peak 31, resulting inI<I′. In case of FIG. 7C, with exception of the peak at the origin, thefirst peak 31 will be eliminated together with the second peak 32, thenonly the noise 35 will increase, resulting in I>I′. The first peak 31may be determined based on the comparison of the result of analysis withand without exception of the peak at origin. FIG. 8A shows the flowchart of the peak identification process. In this flow chart theintensities of the peak of maximum intensity with and without exceptionof the peak at the origin, namely the correlation values will becompared, and the result of analysis without the exception of the peakat the origin will be selected if the correlation value decreases by theexception of the peak at the origin, while on the, other hand theanalysis result with the exception of the peak at the origin will beselected if either the correlation value increases or remains.

[0059] As an alternative, assuming that there are two peaks in theanalysis image P (n, m), the algorithm may be predefined such that thepositions and intensities of two peaks will be output. The lower limitof the correlation should be redefined because the correlation value ofeach of peaks becomes weaker if there are two peaks. FIG. 8B shows twoflow charts of peak identification processes. In cases of FIG. 7A andFIG. 7B the peaks larger than the lower limit of correlation value arethe first peak 31 and the second peak 32, so that two peaks will beoutput. Since |D1|>|D2|=0, when selecting the peak of largerdisplacement from within the output peaks, the first peak will beselected. In case of FIG. 7C since the first peak and second peak areoverlapped, there is only one peak in the P (n, m). If there is only onepeak which is larger than the lower limit of correlation value, thenthat peak will be selected as the analysis result.

[0060] With respect to the setting of tilt angle α, when determining theamount of defocus F based on the amount of displacement D using theparallax, the tilt angle α of electron beam incident to the specimenwill be used, so that the tilt angle α should be set accurately. Themeasurement of the tilt angle α may be accomplished by using thediffracted image of a crystalline specimen having a known diffractiongrid coefficient, such as Au and Si. As the diffraction grid coefficientis already known, if the wavelength of the incident electron beam isdetermined the diffused angle for each pixel in the diffraction imagemay be calculated. The actually measured value of the tilt angle α ofincident electron beam may be obtained by determining the displacement Dα in the first diffraction image taken at the first incident angle withthe second diffraction image taken at the second incident angle tocompute the product of the diffused angle for each pixel with thedisplacement D a. The tilt angle α of incident electron beam isapproximately proportional to the current value IBT flew through thedeflective coil for condenser system 13, however, as shown in FIG. 19the deflective coil for condenser system 13 is mounted above theobjective lens 14 so that the field caused by the objective lens 14 mayalter the angle incident to the specimen. Therefore the compensator itemshould be introduced into the formulation of the tilt angle α ofincident electron beam, using as a parameter the exciting current valueI_(obj) of the objective lens 14. For instance, the formulaα=A*I_(BH)+B*I_(obj)*I_(BH) may be used, where A and B are constantsintrinsic to the apparatus.

[0061] With respect to the amplitude of the tilt angle α, the larger thetilt angle α is, the smaller the amount of defocus F corresponding tothe displacement D of image, therefore the improvement of the precisionof analysis of the amount of defocus F may be estimated. However, thedecrease of common area in the pair of images may invoke a malfunction.The correlation decreases if the common area decreases to the half ofentire image or less, and the reliability of analysis results therebyextremely degrades. Thus the displacement D caused by the parallax isrequired to be set to be less than the half of the length of a side ofCCD camera. When the estimated range of defocus is wider at the samemagnification rate, namely in case of coarse focusing, the tilt angle αshould be set to smaller, whereas when the estimated range of defocus isnarrower namely in case of fine focusing, then the tilt angle α shouldbe set to larger. For example, in case in which the estimated range ofdefocus is 20 microns, the length of a side of electron beam detector is2 centimeters, and magnification rate is 50,000, then the angle α needsto be not more than 0.5 degree.

[0062] It may be possible that the analysis is unavailable because thecommon area of a pair of images is small due to the amount of defocus Fthat was larger than that was estimated. In order to address such acircumstance, a flow process should be provided in which a lower limitof the peak intensity should be set and then the magnification rateshould be lowered if the computed peak intensity goes down below thelower threshold to increase the percentage of the common area tocompensate for the focus in advance to thereby decrease the amount ofdefocus F to restore to the original magnification rate to measureagain. As an alternative in order to increase the common area the tiltangle α may be decreased.

[0063] In the TEM observation the objective aperture to be inserted tothe optical axis are often used to enhance the imaging contrast. It ispossible that, if the direction of incidence of electron beam ischanged, the electron beam will be out of optical axis so that the beamwill not pass through the aperture. In order for the electron beam offirst incident angle as well as the electron beam of second incidentangle to pass through the aperture, the tilt angle α should be setsmaller than the diameter of opening aperture. For instance, for theopening up to 10 microns, the tilt angle α should be set to 0.5° orless.

[0064] Since the second TEM image are to be taken with the incidentelectron beam slanted, if the tilt angle α of the incident electron beamis excessively larger then the image will be distorted due to theinfluence of the eccentric axis, the distortion will cause an extremedecrease of the common area with the first TEM image, resulting in thatthe analysis will be unavailable. In such a case the tilt angle α shouldbe set again to smaller value.

[0065] The magnification rate M also is required for the calculation ofdefocus F. In the conventional TEM there are approximately 5% ofmagnification error.

[0066] In addition, when an optic lens is installed at the photo coupler72 connecting the scintillator 71 to the CCD camera 73, themagnification error of optic lens also should be considered. Nowconsidering the influence of such an error with respect to the errorrate of focusing analysis in case in which there is an amount of errorof M (1+Δ), i.e., Δ in the magnification rate. It is assumed that forexample a displacement D1 was measured. The pure amount of defocus F1may be given by D1/ /[M (1+Δ) α]⁻¹−Cs α², but the amount of defocus F1′may be given by D1/[M α]−Cs α². The error of analysis of defocus Fcaused by the magnification error then may be F1−F1′−ΔD1/(1+Δ)Mα. Thefocusing error caused by the magnification error may be proportional tothe amount of displacement D1. In other words, when the displacementD=0, the focusing error caused by the magnification error will be thesmallest. Then the focus compensation toward Fs=−Cs α² where thedisplacement D=0 may be attempted. When attempting to set the focus asFs after the amount of defocus F1′ has been determined, the focus willbe (F1−F1′)+Fs. The displacement D2 at this stage will be measured asD2−ΔD1. Since the magnification error A of the TEM is approximately 5%,a few times of focus compensation may lead to the displacement D ˜=0. Inthis manner, it can be seen that the focus error caused by themagnification error will be sufficiently small by means of such aprocess flow that the optimum focus specified may be set after theobjective lens has been tuned to the displacement D=0. The process flowmay alternatively be such that the focus is adjusted to D=M Cs α³, whereF=0 in the vicinity of D=0, not at the displacement D=0. In this casethe influence of magnification error will be decreased together with theinfluence of the second peak caused by the fixed pattern. Thefunctionality is added which may abort the compensation for focusing incase in which the repetition of compensation for defocus F=0 is morethan two and the amount of defocus Fn determined at nth path is largerthan the amount of defocus F_(n−1) determined at n-1th path. This allowsthe repetition of compensation to be held to the minimal requirement.

[0067] By taking above into consideration, focusing will be performed inaccordance with the flow chart shown in FIG. 9. At the beginning, afield of view on which focus will be analyzed will be selected. Theselection of a field of view includes also the setting of magnificationrate of the observation and of objective aperture. Then the displayexamples shown in FIG. 2A and 2B will be used to set the optimal focus,lower correlation threshold, tilt α,and repetition of compensation. Therecommended values i.e., the initial default values of the optimalfocus, lower correlation threshold, tilt angle α, and repetition ofcompensation are preset in the computer, which may be changed by theoperator when required. Although the optimum focus is usually set toF=0, there may be cases in which under focus observation may bepreferable depending on the specimens. The tilt angle α is set atdefault to 0.5°, the largest angle α available for passing the electronbeam through the objective aperture of diameter up to 10 microns,however, there may be cases in which the tilt angle should be set to asmaller value, because the image distortion caused by the change ofelectron beam incident angle may severely affect to a certain fields ofview. The lower correlation threshold also depends on the photographicconditions such as the number of pixels of the analysis image and thelike. In order to optimize the tilt angle α and the correlation value,it will be necessary to provide a mode in which focusing will beanalyzed but not compensated. In general the tilt angle α used routinelywill be in the range from 0.2 to 0.5°. The lower limit of the tilt angleα and the upper limit of precision tolerance may be determined by theperformance of the system. Only the measurement may be performed bysetting the repetition of compensation shown in the display screen ofFIG. 2 to 0, and clicking the button 93 for repeating focusing. If theoperator cannot recall the recommended initial values during setting ofthe parameters, then by clicking on the “initialize” button 92, allparameters will default to the initial values.

[0068] Once the parameters are set, a pair of images may be taken byusing the electron detector 17. The conventional focus detectors of TEMoscillates sinusoidally the angle of incident electron beam by using thedeflective coil for condenser system 13 and the operator observes thevibration of TEM image.

[0069] Since the TEM image continues to vibrate in the conventionalconfiguration of circuitry, capturing of images is not available. Inorder to capture images, a control system is required in which an imageis captured upon receiving a signal instructing the capture of first TEMimage, then the incident angle of electron beam is changed to secondangle upon receiving a signal indicating the image capture has beencompleted, and a second image is captured thereafter. From a pair ofimages captured by using such a control system, an analyzing image P (n,m) will be computed to identify the peak corresponding to thedisplacement.

[0070] If any peaks corresponding to the displacement cannot beidentified, a flow is provided so as to abort the focusing operation andto estimate the cause for instructing the operator what to do next. Thepossible causes which may fail the analysis of displacement may becaused by a problem in the image, such as for example no specimen foundin the field of view, and an image extremely out of focus, or a problemof decrease of the common area between images in the pair such as theamount of displacement D excessively large due to the inclination toolarge of incident electron beam angle, and the distortion of imagesaffected by the change of incident electron beam angle. In order todetermine the cause, a fourth TEM image will be captured by translatingthe image by a known amount of distance in a direction by using thedeflective coil for condenser system 16 at the first angle of incidentelectron beam to analyze the displacement between the first TEM imageand the fourth TEM image. If, as a result, displacement analysis isimpossible between the first and fourth TEM images, then there is aproblem with respect to the images such as no specimen in the field ofview, or the image extremely out of focus, or the like. To resolve sucha problem an instruction may be issued so as to decrease themagnification to perform a preliminary focusing at lower magnification.At a lower magnification a vaster field of view may be obtained,resulting in that the chance of finding the specimen in the field ofview will increase. In addition at a lower magnification the influenceof blurred image caused by the defocus may be decreased and thesharpness may be improved so that the analysis of displacement will beenabled. If the displacement between the first TEM image and fourth TEMimage may be analyzable, the cause may be the decreased common areabetween images in the pair, therefore an instruction should be issued toreduce the amount of tilt angle a. The system will display an errormessage on the display screen as shown in FIG. 2(b). Although thecorrelation rate will be displayed the amount of defocus F that couldnot be determined will not.

[0071] Once the peak corresponding to the displacement has beenidentified, the current through the objective lens will be adjusted bydetermining the displacement D therefrom to calculate the amount ofdefocus F by means of the relation D=α(F+Cs α²).

[0072] Since the relationship between the current of objective lens andthe focal distance may vary depending on the parameters of the projectorlens that means the observing magnification rate, a relational table ora relation describing the current of objective lens with the focal pointfor each observation magnification rate is stored in the computer. Thecurrent of objective lens will be adjusted by using the table orrelation to determine the current required for focusing at the desiredfocus. In case in which the number of repetition of focusing is set totwo or more, another pair of images will be taken to analyze the focusto readjust the current of objective lens.

[0073] The automatic focus compensator system in accordance with thepresent invention incorporates a displacement analysis processor usingphase variance of Fourier transform images 20, implemented by a digitalsignal processor (DSP), which may complete the analysis of displacementof an image of 256 by 256 pixels within 30 milliseconds, in comparisonwith the same calculation for 2 seconds by a conventional applicationsoftware. One focus compensation cycle will be completed within onesecond, including the image capture by the electron detector 17, theadjustment of the deflective coil for condenser system 13 and theobjective lens 14, and the system can also perform an iterativefocusing. The iterative focusing starts by clicking the button to repeatfocusing, as shown in FIG. 2, and the iterative focusing stops byclicking the button to stop focusing. Alternatively by clicking thebutton to repeat focusing the iterative focusing starts, and thefocusing may be stopped by clicking the same button again. Alternativelyby double clicking the button to execute focusing 93 the iterativefocusing starts, and the focusing stops by clicking the button toexecute focusing 93 again. In the course of repeating focusing the focuswill be automatically readjusted to an optimal focus even when the fieldof view changes by moving the specimen stage. When the first TEM imageand the second TEM image are taken during moving the specimen stage, theprecision of focal analysis will be degrade because of introduction ofthe displacement D caused by the parallax along with the displacement Dsof moved specimen stage, however, the precision of focusing required forthe specimen observation may be obtained since the observation may bestarted by stopping the specimen stage when the desired field of viewhas been found. In case in which the degraded precision of compensationby the moving specimen stage is concerned, the focus may be analyzed bymeasuring the moving speed of the specimen stage from the displacementbetween the first TEM image captured for the nth focusing analysis andthe first TEM image captured for the n-1th focusing analysis to estimatethe displacement Ds caused by the moved specimen from the first TEMimage to the second TEM image in the nth measurement, and by subtractingthe displacement Ds caused by the moved specimen from the displacementD+Ds between the first TEM image and the second TEM image to extract thedisplacement D caused by the parallax.

[0074] In the present system a malfunction checking functionality isbuilt in so as to hold the focus setting if the correlation value isless than the lower threshold. The TEM image in general has low SINratio, and the image of low S/N ratio may potentially have a higherprobability that the displacement analysis is not executable. If thedisplacement analysis is unavailable because of the probability, thenthe analysis in the next turn will have a higher probability ofobtaining a correct result. Therefore the upper limit of the number oferrors will be predefined in the focus analysis. If the number of timesthat the correlation falls below the lower threshold exceeds the upperlimit, the system provides a functionality to determine whether therehas been an accidental event, such as the TEM image of the structure ofspecimen was not captured by the electron beam detector because theelectron beam was blocked by for example the aperture and the like, andto display an error message on the screen.

[0075] During the iterative focusing, the first TEM image by the firstincident angle and the second TEM image by the second incident angle arealternately displayed on the display screen. When the image processingspeeds up, the alternate display may be perceived as a kind of flickerof the display, giving the operator an uncomfortable feeling or adifficulty to observe fine structures. Therefore the present systemprovides a configuration in which the TEM image captured with the firstincident angle will be displayed on the screen, whereas the TEM imagecaptured with the second incident angle will not be displayed. Anotherconfiguration may also be provided, in which the TEM image observed atthe first incident angle and the TEM image observed at the secondincident angle are separately displayed, so that the influence of imagedistortion caused by the axial displacement of the incident electronbeam may be confirmed when required.

[0076] [Second Embodiment]

[0077]FIG. 19 shows a fundamental arrangement of TEM used in anautomatic analyzer. The TEM is comprised of an electron gun 11 andelectron gun control circuit 11′, a condenser lens 12 and condenser lenscontrol circuit 12′, a deflective coil for condenser system 13 anddeflective coil control circuit for condenser system 13′, an objectivelens 14 and objective lens control circuit 14′, a projector lens 15 andprojector lens control circuit 15′, a deflective coil for condensersystem 16 and deflective coil control circuit for condenser system 16′,an electron detector 17 and electron detector control circuit 17′, aspecimen stage 18 and specimen stage control circuit 18′, and a computerwith control software and image processing software 19. Each of controlcircuits may receive control commands sent from the control software inthe computer 19, perform controls and return the return value to thecomputer. There is provided an image shift function for translating theTEM image by using the deflective coil for condenser system 13 and thedeflective coil for condenser system 16. The electron detector 17 is adetector constituted of a plurality of pixels such as a CCD camera,which may transmit signals of obtained images at a higher rate throughthe cable for image transmission to the storage device of the computer19 or to the displacement analysis processor using phase variance ofFourier transform images 20. The displacement analysis processor usingphase variance of Fourier transform images 20 is connected to thecomputer with control software and image processing software 19, whichfurther comprises a software for pattern inspection and measurement.

[0078]FIG. 10 shows a process flow of the automatic analyzer using theTEM in accordance with the present invention. An acceleration voltage isapplied to the electron beam generated by the electron gun 11, then thedeflective coil for condenser system 13 is used for adjusting thedeflection of beam such that the electron beam passes through theoptical axis, to verify that the electron beam reaches to the electrondetector 17. In this document ‘z’ axis is defined as an axis in parallelto the optical axis, x-y plane is defined as the plane normal to theoptical axis. After adjusting the condenser lens 12, a specimen 21 isput into the specimen chamber. The specimen 21 is thinned (sliced) toallow the electron beam to pass through, and then is mounted on ametallic support member that is also called a ‘mesh’ 22 (FIG. 11(a)).The mesh 22 is placed in a specimen holder, which is in turn placed onthe specimen stage to be observed. Since the diameter of the mesh 22 isapproximately 3 millimeters, it is difficult to accurately specify thedirection and position thereof when placing on a specimen holder.Therefore in accordance with the process flow shown in FIG. 12 the imageof the mesh 22 observed at a lower magnification rate is recorded toanalyze the direction, position, and shape of the mesh 22. The electronsmay transmit only through the void region called ‘hole’ 23. At first,the captured image will be binary coded to determine the connectivecomponents to label each region (see FIG. 11B). Then the surface area ofeach of labeled regions will be calculated. Since the holes are almostconstant in size, the modal (most frequent) value among the surface areavalues may be defined as the surface area of a hole. More specifically,among the regions labeled as shown in FIG. 11B, the regions having asize in proximity of the modal are regions labeled as 4, 5, 7, 8, 9, 12and 13, where the shape of entire holes is depicted. To analyze thedirection of the mesh 22, the center of gravity 24 of the labeledregions in which the hole 23 is entirely depicted will be computed. Thena combination having the shortest distance between the center of gravityof a region with respect to another will be determined (FIG. 1C). Forinstance, the nearest centers of gravity from the center of gravity 24of the labeled region 4 are the centers of gravity 24 of the labeledregion 5 and of the labeled region 8. The direction connecting thecenter of gravity 24 of the labeled region 4 with the center of gravity24 of the labeled region 5 will be defined as the x direction, while thedirection connecting the center of gravity 24 of the labeled region 4with the center of gravity 24 of the labeled region 8 will be defined asthe y direction. Since the shape of the hole 23 is known, the height Hof a side of hole 23 may be given from the surface area of the hole 23.By subtracting the height H of a side of hole 23 from the span of theshortest line between the nearest centers of gravity, the margin M ofthe mesh 22 may be computed. Once such calculation is completed, animage display that the x-y direction of the mesh 22 matches with thehorizontal and vertical direction of the display will be displayed. Eachhole 23 of the mesh 22 will be numbered. The operator may confirm onthis display at this point that the labeling has been correctlyexecuted. By directing a number labeled to a hole 23, a hole 23containing a specimen 21, i.e., a hole 23 to be inspected may be definedso as not to inspect other holes to shorten the time of inspection.

[0079] The presence or the absence of specimen in a hole may bedetermined automatically by image processing. The automatic decisionuses a histogram or Fourier transform image of the image intensity inthe hole. The decision using a Fourier transform image uses thepercentage of high frequency component in the Fourier transform image.

[0080] When no specimen is present in the analytic area, as shown inFIG. 17B, the Fourier transform image contains only low frequencycomponents, even though there is a certain fluctuation in the imageintensity depending on the distribution of the current density ofirradiated electron beam. If a specimen is present which contains a finestructure such as in case of biological specimens, the percentage ofhigh frequency in the Fourier transform image will be increased (FIG.17A). It may be determined that the specimen is present in the analyticarea to be inspected in case in which the percentage of high frequencycomponent with respect to the low frequency component exceeds a certainthreshold. When determining based on the histogram of image intensity,the presence or absence of specimen will be determined in accordancewith the number of peaks present in the histogram of the intensity ofimage. If no specimen is present in the hole, there will be only onepeak (see FIG. 13B) even though the image intensity may fluctuate moreor less depending on the distribution of the current density ofirradiated electron beam. On the other hand if there is a specimen inthe hole, then there will be a plurality of peaks present in thehistogram of image intensity containing the specimen in the hole (seeFIG. 13A). Accordingly it may be determined that the specimen is presentin the hole when there is a plurality of peaks in the histogram of imageintensity. It should be noted that the presence or absence of specimenmay be determined based on the half-width of peaks in case of specimenswhich have a contrast so low that the peaks may be overlapped.

[0081] Prior to analyzing the direction and the shape of mesh 22, it maybe desirable to adjust the height of the specimen holder. There may be acase in which the specimen holder is placed outside the focusing rangethat the objective lens maybe adjust, because of inappropriateadjustment of the specimen stage 18. When the stage is appropriatelyplaced within the focusing range, the magnification rate and the likemay vary due to the variation of lens condition when the current flewthrough the objective lens is drastically varied, therefore it may bedesirable to keep the height of the specimen holder approximatelyconstant. In the focusing analyzer as will be described later, afunctionality is provided to automatically adjust the height of thespecimen holder by analyzing the height of the specimen holder anddriving the specimen stage control circuit 18′.

[0082] The specimen holder may be inserted slantly because of forexample inappropriate setting of specimen stage 18. If the specimenholder is seriously inclined, the condition of observation depends onthe position of hole in the mesh 22. Therefore by selecting a pluralityof points in the mesh 22, the position of those points and the height ofspecimen determined by the analysis of defocus will be recorded. Theinclination angle of the specimen holder may be given by the height ofspecimen in each of positions, and the inclination of the specimenholder can be adjusted accordingly.

[0083] The specimen stage control circuit 18′ contains the automaticadjuster of the inclination of the specimen holder.

[0084] Then items of inspection will be set. Most of automaticinspection of biological specimen are often the research of the shapeand number of viruses. For each of viruses various items of inspectionsuch as preprocess of image and the geometrical characteristics to besearched may be configured and stored in the computer 19 as a macroprogram. For instance, now considering a case of measuring the number ofspherical viruses having diameter in the range of approximately 20 nm to30 nm, dispersed in the specimen, and the diameter thereof. The items ofinformation required to be inspected are solely the number and diameterof viruses, then the TEM image of the specimen having viruses stronglystained should be captured by using the electron detector 17, then theimage thus captured should be binary coded to extract the stainedregions, i.e., viruses to analyze the geometrical characteristics tomeasure the diameter of viruses. To this end, the size of the area to beanalyzed will be selected. The number of pixels contained in an imageused for the viral inspection may be 512 by 512 pixels, and whenmeasuring the diameter of viruses within an error of approximately 10%,in order for the diameter of a virus to be about 10 pixels the suitablelength of a side of square of the analytic area will be preferably about1 micron. If the length of a side of hole is 30 microns, then one holewill be divided into 30 by 30 areas, or 31 by 31 areas with an overlapof about 30 nm between areas so as to prevent the count from beingdropped therebetween. Once the division of areas has been completed adisplay as shown in FIG. 11D will be displayed on the screen to indicatethe areas to be inspected.

[0085] Since every areas to be inspected do not contain a specimen, ifthe specimen is not present in an area to be inspected, it will bebetter move to next area immediately for saving the time of inspection.The system provides a functionality of determining the presence orabsence of the specimen in a specific area to be inspected, by using thehistogram of image intensity or the Fourier transformed image in thatarea. As have been described above, the specimen was prepared such thatonly viruses are strongly stained, the area containing the specimen maybe presumably different in the image intensity from the area containingno specimen. If there is the specimen present in the area to beinspected, as shown in FIG. 13A, the histogram of the image intensitywill have a plurality of peaks. On contrary if the specimen is notcontained in the area to be inspected as shown in FIG. 13B, then therewill be only one peak, though the image intensity may fluctuate inaccordance with the distribution of the current density of irradiatedelectron beam. As a result, the presence or absence of viruses may bedetermined in accordance with the number of peaks present in thehistogram of image intensity. In case of the decision using the Fouriertransformed image the decision will be made based on the percentage ofhigh frequency component in the Fourier transformed image. If thespecimen is not present in the area to be inspected, as shown in FIG.17B, there is only low frequency component in the Fourier transformedimage. On the other hand, if the specimen is present in the area to beinspected, which contains a fine structure such as in case of biologicalspecimen, the percentage of high frequency component in the Fouriertransformed image will be augmented (see FIG. 17A). It may be determinedthat a specimen is present in the analytic area in case in which thepercentage of high frequency component with respect to the low frequencycomponent exceeds a certain threshold.

[0086] In a hole of 30 microns square, the diagonal distance is 42microns. If the specimen holder is inclined by 1°, a defocus of 0.74micron may be occurred in the hole. The contrast of TEM images issusceptibly affected by the defocus. If there is a defocus in the orderof submicron, the image will be blurred or the contrast will be varied.Viral inspection has to be always performed with the image taken at aconstant and precise focus. The focusing should be well compensated forin the order of submicron prior to starting a viral inspection.

[0087] The focal analysis using the parallax is applied to the focalanalysis in such a focus compensation. A first TEM image taken with theelectron beam incident from a first angle approximately in parallel tothe optical axis and a second TEM image taken with the electron beamincident from a second angle inclined by a tilt angle α from the opticalaxis are used. As shown in FIG. 4, if the focus is not to the point,there may be a certain displacement of image between the first TEM imageand the second TEM image. The defocus F and the displacement D caused bythe parallax are related in D=M α(F+Cs α²). The magnification M and thetilt angle α may be selected by the operator. The spherical aberrationcoefficient Cs is intrinsic to the apparatus, so that the defocus F maybe identified if the displacement D between images in the pair isdetermined. The conventional method of analysis of displacement usedheretofore such as cross-correlation method, least-squares method andthe like, could not obtain a sufficient precision of focal analysisbecause the analytic precision of the analysis method of displacementdid not reach to less than one pixel. The present invention ischaracterized in that it applies a method of analysis based on the phasevariance analysis of the Fourier transformed image to the analysis ofthe displacement D. As shown in FIG. 1, the first and second TEM imageshaving the incident angle of the electron beam varied with respect tothe specimen by using the deflective coil for condenser system 13mounted above the objective lens 14 will be captured by the electrondetector 17. Thus captured first and second TEM images will betransmitted to the displacement analysis processor using phase varianceof Fourier transform images 20 and in turn the displacement D that isthe analytic result will be transmitted to the computer 19, which willcompute the amount of defocus F from the displacement D to determine thecurrent of the objective lens I_(obj) required to adjust the focus tothe target point, and then compensate for the focusing of the objectivelens 14.

[0088]FIG. 5 shows a schematic diagram illustrating the displacementanalysis method applied to the present invention. Now assuming a pair ofimages S1 (n, m)=S2 (n+dx, m+dy) having a certain displacement D=(dx,dy), and the two dimensional discrete Fourier transform of S1 (n, m) andS2 (n, m) to be S1′ (k, l), and S2′ (k, l). From the formula F {S (n+dx,m+dy)}=F {S (n, m)} exp (idxk +idyl) of the Fourier transform, S1′ (k,l)=S2′ (k, l) exp (idxk+idyl) may be obtained. The displacement in S1′(k, l) and S2′ (k, l) above may be expressed by the phase variance exp(idxk+idyl)=P′ (k, l). P′ (k, l) is a wave with the cycle (dx, dy), thenin an image P (n, m) which is subjected to invert Fourier transform of aphase variant image P′ (k, l), a δ peak will be appeared at the location(dx, dy) Since it can be assumed that in the image P (n, m) only the δpeak may be present, the position of δ peak may be given by thecomputation of the center of gravity of the intensity of δ peak even ifa fraction is present.

[0089] The intensity of δ peak calculated after normalizing theintensity of entire image P (n, m) will be weaker if the noise i.e.,discrepancy between images in the pair is increased. Thus the operatormay identify the signal-noise ratio, i.e., reliability of the analysisresult by indicating the peak intensity as the correlation value. In theautomatic compensator the analysis is not always assured to be accuratein every area. Therefore, the lower threshold of the correlation will bepredetermined so as no to adjust the objective lens if the correlationvalue calculated is below the lower threshold to record the address ofthe analysis area along with the correlation value. For example, if theposition of mesh is shifted out of the point by an incorrect operationin the course of transfer of the specimen stage, more than half of acaptured TEM image will be occupied by the mesh 22 as well as the commonarea of the pair of images will decrease so that a sequence ofunanalyzable area will be left at the edge of holes 23. Once thedisplacement D has been analyzed, after the automatic inspectioncompleted, to allow to determine, based on the distribution ofunanalyzable area, whether the mesh was moved into the analysis area dueto the inaccurate operation of the specimen stage and at which step inthe course the incorrect transfer of the specimen stage was occurred,the operator may instruct to compute the focus F using the relation D=Mα(F+Cs α²) to determine the current value required for bringing to thespecified optimal focus to adjust the current of the objective lens.After the objective lens is adjusted, by performing another focusanalysis using the parallax to record the correlation value in thisdisplacement analysis and the current of the objective lens along withthe address of the analysis area, the status of the inspection may berecorded in greater details. The distribution of the specimen height maybe derived from the current of the objective lens set at the optimumfocus. In addition the image quality such as sharpness may be allowed tocompare by using the correlation value calculated at the samedisplacement D.

[0090] Once the focusing adjustment has been completed, a viralinspection may be started in accordance with the process flow shown inFIG. 14. Although another TEM image may be taken for the inspection, aTEM image which has been already taken at the incident angle 1 of theelectron beam with the optimum focus is recorded, and this image may beused for the viral inspection in order to save the time of imaging. In aviral inspection, the TEM images will be binary coded to make theconnecting component to label every regions. Then, the surface area ofeach of labeled regions will be computed to eliminate the region havingless than a predetermined surface area, because these smaller regionsmay be determined as noise. Then, the characteristic amount ofbiological specimen will be computed from the roundness and moment ofeach of the labeled regions, and the region identified to be closer to aroundness will be determined to be likely a virus, thereby the diameter(biological information) will be derived from the surface area. Thenumber of viruses and the diameter of each virus will be recordedtogether with the address of the analytic area in a similar manner.

[0091] Once the inspection has been completed in one area to beanalyzed, the specimen stage 18 may be used to transfer the specimen tomove to a next area to be inspected to start the inspection. Theprecision of fine adjustment of the specimen stage 18 may be describedby the registering accuracy and the back-rush thereof. The registeringaccuracy is the accuracy of transfer in a constant direction of movingthe specimen stage, the back-rush is the distance of slipping at thetime of turning the direction. In the products currently available inthe market, the registering accuracy is achieved in the order of about1.2 nm, back-rush in the order of about 0.02 micron. In case of analyticareas of diameter of 30 microns, the specimen transfer may beaccomplished by using the specimen stage 18.

[0092] However, when moving the specimen stage 18, a certain amount ofspecimen drift may be occurred by the inertia of the specimen stage 18.In the focusing analysis using the parallax, the precision of focusinganalysis will be degraded if the displacement D caused by the parallaxis intermixed with a certain amount of displacement Ds caused by thedrift of specimen. To avoid this, a third TEM image, which may be takenat the first incident angle of the electron beam at a second timedifferent from the time at which the first TEM has been taken will beused. The amount of specimen drift may be computed from the amount ofdisplacement between the first TEM image and the third TEM image. Thisdisplacement analysis also may be performed by means of the displacementanalysis using phase variance of Fourier transform images. The precisionof focus analysis is likely to be degraded unless the displacement Dscaused by the specimen drift is analyzed at the same analytic precisionas the analysis of displacement D. In addition, the measurement of driftof specimen has to be completed within a very short period of time, andthe amount of displacement caused by the specimen drift are to besignificantly small. The conventional analysis of displacement, in whichthe analytic precision may be limited by the size of a pixel, obviouslyhas not sufficient precision. The displacement D caused by the parallaxmay be given by subtracting the displacement Ds caused by the specimendrift from the amount of displacement between the first and second TEMimages. In addition, the blur caused by drift in the captured images maybe removed by performing an automatic drift compensation for operatingthe deflective coil for condenser system 16 so as to cancel thedisplacement Ds caused by the specimen drift.

[0093] The occurrence of specimen drift which may affect to the focalanalysis may be estimated in some extent, such as at the time when theelectron microscope has been just powered on, during the period of timeuntil the difference of temperature in the microscopy and/or theelectron gun will be settled, and at the time immediately after themoving specimen stage 18 is stopped. Since the efficiency of analysiswill be lowered if a number of images are to be captured, then thecondition of observation to capture the third TEM image for compensatingfor the drift may be predetermined, and when the condition matches, thethird TEM image will be taken along with the first and second TEM imagesso as to enable to eliminate the influence of drift. If the algorithm isimplemented in which only the first and second TEM images are capturedonce the drift decreases, in other words when the amount of displacementbetween the first TEM image and the third TEM image reaches to or in thevicinity of zero, the accurate compensation of focusing may beaccomplished with the least number of TEM images required.

[0094] The efficiency of inspection will be degraded if a third TEMimage used for compensating for the drift of specimen is taken each timethe specimen stage 18 moves. Therefore, the transfer of the specimenstage 18 may be limited to the transfer between holes and the transferbetween the analytic areas may be performed by the deflective coil forcondenser system 16. The number of third TEM image to be taken will besignificantly decreased since the compensation of specimen drift causedby the inertia of the stage transfer will be performed only when movingbetween holes. Other examples requiring the transfer of analytic area byusing the deflective coil for condenser system 16 include, for example,a case in which the final precision is insufficient with the precisionof fine focus adjustment of the specimen stage 18 with respect to thetransfer of analytic area because the analytic areas are subdivided intosmall areas.

[0095] If the size of a hole is enough vast so that the deflective coilfor condenser system 16 cannot follow, the image may be shifted bymoving the specimen stage 18 into a direction at a constant velocity andusing the deflective coil for condenser system 16 to move at anapproximately same velocity. FIG. 18 C shows the location of the fieldof view when the transfer of the specimen stage is used together withthe image shifting in order to move between analytic areas, theparameters of the deflective coil for condenser system 16, and theposition of the specimen stage 18 along with the elapsed time. Here Tcdesignates to the time required for taking a TEM image, Ts to the timerequired for transfer of the field of view by the specimen stage, and Tito the time required for the transfer of the field of view by thedeflective coil for condenser system 16. The transfer of the field ofview by using the specimen stage 18 may be accelerated but the speed-upis limited due to the influence of inertia when moving the stage and theback-rush, the transfer of field of view by using the specimen stage 18may not be accelerated faster than the transfer of field of view byusing the deflective coil for condenser system 16. If the field of viewis transferred solely by means of the specimen stage 18, the time ofinspection will be prolonged as shown in FIG. 18A. On the other hand thedeflective coil for condenser system 16 has a disadvantage of narrowerrange of transfer. As shown in FIG. 18B, when the transfer is out ofrange of the deflective coil for condenser system 16 the specimen stage18 should be used to move the specimen. Accordingly, when moving thespecimen stage 18 at a constant velocity as shown in FIG. 18 C whileusing the image shifting by means of the deflective coil for condensersystem 16 so as to cancel out the movement of the specimen stage, stillimages of each of analytic areas may be taken even when the specimenstage 18 is in the course of transfer. In this manner the transfer ofthe field of view in a vast range may be carried out at high speedwithout influence of the backrush and inertia of transfer of thespecimen stage 18.

[0096] The analysis may be performed in a vaster range if the positionof the deflective coil for condenser system 16 is approximately constantat the moment of the beginning of inspection for each analysis area (seeFIG. 18). To do this, the transfer velocity of the specimen stage 18should be set, by calculating the time window T required for both thecompensation and inspection carried out in each analysis area such thatthe transfer to the next analysis area by means of the specimen stage 18may be completed before this time window T expires. The amount of imageshifting by means of the deflective coil for condenser system 16 will bemanaged so as to be able to cancel thus decided transfer speed of thespecimen stage 18. In order to match the transfer speed of specimen bymeans of the specimen stage 18 with the shift speed of the deflectivecoil for condenser system 16, the analysis of displacement in the fieldof view may be performed by the displacement analysis using phasevariance of Fourier transform images. As shown in FIG. 26, after settinga first time and a second time, a first TEM image at the first time aswell as a third TEM image at the second time will be captured by usingthe electron detector 17. Thus captured first and second TEM images willbe transmitted to the displacement analysis processor using phasevariance of Fourier transform images 20, from which the displacement Dresulted by the analysis will be further sent to the computer withcontrol software and image processing software 19. The computer 19 willcompute the moving velocity of the field of view based on thedisplacement D to determine the parameters of deflective coil forcondenser system 16, which are required for the transfer speed of thefield of view to be zero, and to adjust the deflective coil forcondenser system 16 based on the parameters thus determined.

[0097] It is preferable to keep constant the time T of inspection foreach analysis area, since the positional precision of the specimen stage18 is higher when the transfer speed of the specimen stage 18 isconstant. Thus it is preferable to keep constant the number of images tobe taken for each analysis area. Otherwise if a third TEM image foranalyzing the displacement of the analysis area is taken for each ofanalysis areas, the precision of displacement compensation and theprecision of the focusing analysis may be improved, whereas theefficiency of inspection deteriorates. Accordingly, The transfer of thefield of view by using the specimen stage 18 as well as the transfer byusing the deflective coil for condenser system 16 will be adjusted inthe stage of adjusting the microscope prior to the viral inspection.Alternatively any analytic areas inappropriate for the viral inspectionmay be predefined so as not to perform focusing in the areas, but thefirst TEM image and third TEM image may be taken to adjust the transferof the field of view. By assuming that the field of view in the usualanalysis areas will be virtually stationary, the first TEM image andsecond TEM image will be taken for adjusting the focus.

[0098] The items displayed in the course of inspection may be selectedby the operator as required. For example, items as shown in FIG. 15 maybe displayed on the screen. On the screen, the image of the analysisareas into which the image of the mesh taken 22 are divided will bedisplayed in a window, in which the analysis area that the inspectionhas been completed, area that the inspection is in progress, and areathat is not yet inspected will be displayed in different colorsrespectively, in order for the operator to be able to make use thereoffor the understanding of the progress of the inspection and for theestimation of the time of completion. There may be provided also awindow showing a table 94 for sequentially displaying the result ofanalysis in each of the analysis area, and a histogram 95 for displayingthe cumulative values of the results. Also a window for displaying theresult of focal analysis may be provided, which may display the heightof specimen calculated from the correlation value between the pairedimages and from the result of focal analysis. For the reference point ofheight of specimen an appropriate location in the specimen may beselected before or after inspection and that location may be specifiedby clicking the reset button 96. Within a window of TEM image for use ofviral inspection a layer is provided for displaying circles 97 thatindicates the position and the size of viruses identified. The operatorwho checks the results of viral inspection, focusing, and TEM image canabort that inspection if something goes wrong. The items specified bythe operation among the result of viral inspection, focusing, and TEMimage will be stored in the memory so as to allow the operator todisplay, after the inspection, the result of inspection of the analyticareas in which anomalies is prospected to be happened, based on theinformation such as the correlation value and the height of specimen andto confirm the inspection status.

[0099] The mesh 22 is also used for the display of analytic result afterthe inspection. The inspected holes are divided into several areas, andthe result with respect to each area may be displayed as black and whiteor in full color in accordance with the selection of displayed items.For instance, when the operator selects the number of viruses for thedisplay item, each area will be colored in accordance with the number ofviruses, as shown in FIG. 16B. The TEM image taken for each of analysisareas may be shown on the screen by for example specifying the number ofarea in interest, or by double clicking on the spot of the analysisarea. Among items stored in the memory, only the items specified by theoperator will be displayed on the screen. For example, the result ofviral inspection in that area in interest, the height of specimen, thecorrelation value may be displayed along with the TEM image display(FIG. 16A).

[0100] The distribution chart of the height of specimen and thecorrelation may be used for outlining the inspection status and forevaluating the reliability of the inspection result. Since biologicalspecimens are in general sliced into thin sections of thickness of abouttenth nanometers, which may be considered to be almost flat. The changeof the height of specimen may be caused by the curved or inclined meshthat mounts the specimen. When plotting the distribution of the heightof specimen the distribution of height should form a curved surface ofthe kind relatively smooth. If the height of specimen changes abruptly,then it can be concluded that either an incorrect operation occurred inthe focusing analysis of that area, or the sectioned specimen was blownup for some reason. In either case, the reliability of the result ofinspection of the analysis area in question is not sufficient. Forexample, when considering the distribution of the height of specimen asshown in FIG. 16 C, as the height of specimen in the region 25 isdifferent from other regions, it can be concluded that the reliabilityof the result of inspection in the section 25 is not eligible. In orderto remove the result of analysis in this region, the setting may beconfigured so as no to use the result of analysis in any analytic areaout of specified range of the height of specimen. This means that thedistribution of the height of specimen may be used as a sort of filterapplied to the result of analysis. Alternatively, instead of configuringa filter based on the height of specimen, the curvature obtained fromthe distribution of height may be used for configuring a filter. When adistribution of correlation as shown in FIG. 16D has been obtained, thiscorrelation may be also used for the evaluation of the sharpness of TEMimages. In case of biological specimens, if the section of slicedspecimen is thick, even images observed at the optimum focus will beblurred. With blurred images the correlation will be degrades and theerror encountered at the time of binary coding will be larger so thatthe precision of measurement of the diameter of viruses will be lowered.By using the distribution of correlation as a filter, in a similarmanner to the distribution of the height of specimen, the analysisresult in the region 26 having a lower correlation than others may beeliminated. By estimating the measurement error of the viral diameterbased on the correlation values, the measurement error may be used as aweighting function when creating a distribution chart of the diameter ofviruses.

[0101] [Third Embodiment]

[0102]FIG. 19 shows a fundamental arrangement of a transmission electronmicroscope (TEM) for use in an embodiment in accordance with the presentinvention. The TEM is comprised of an electron gun 11 and electron guncontrol circuit 11′, a condenser lens 12 and condenser lens controlcircuit 12′, a deflective coil for condenser system 13 and deflectivecoil control circuit for condenser system 13′, an objective lens 14 andobjective lens control circuit 14′, a projector lens 15 and projectorlens control circuit 15′, a deflective coil for condenser system 16 anddeflective coil control circuit for condenser system 16′, an electrondetector 17 and electron detector control circuit 17′, a specimen stage18 and specimen stage control circuit 18′, and a computer with controlsoftware and image processing software 19. Each of control circuits mayreceive control commands sent from the control software in the computer19, perform controls and return the return value to the computer. Theelectron detector 17 is a detector constituted of a plurality of pixelssuch as a CCD camera, which may transmit signals of obtained imagesthrough the cable for image transmission to the storage device of thecomputer 19 or to the displacement analysis processor using phasevariance of Fourier transform images 20. The displacement analysisprocessor using phase variance of Fourier transform images 20 isconnected to the computer with control software and image processingsoftware 19.

[0103]FIG. 3 shows a flow chart of TEM imaging. An acceleration voltageis applied to the electron beam that is the first charged particle beamgenerated by the electron gun 11, then the deflective coil for condensersystem 13 as a deflector means is used for adjusting the deflection ofbeam such that the electron beam passes through the optical axis, toverify that the electron beam reaches to the electron detector 17. Afteradjusting the condenser lens 12, a specimen 21 is set into the TEM, anda TEM image at lower magnification rate is observed. The objectiveaperture is inserted to the optical axis in order to increase thecontrast of TEM image. By gradually increasing the magnification of theprojector lens 15, an observation field is selected and the focusing isadjusted to take TEM images as required.

[0104] To the analysis of focusing in the above focusing step is applieda focusing analysis method using parallax. In this method a first TEMimage obtained by an electron beam emitted at first incident angle inalmost parallel to the optical axis, and a second TEM image obtained byan electron beam emitted at second incident angle descend to an angle αfrom the optical axis are used. A functionality for deliberatelychanging the irradiating angle of the electron beam into the specimen byusing the deflective coil for condenser system 13 as shown in FIG. 19 iscalled a wobbler, which may be used for transform an amount of defocusinto a parallax. Now referring to FIG. 24, the principal of operation ofthe wobbler and the mechanism of occurrence of parallax will bedescribed below in greater details. In FIG. 24(a) there is shown anoptical geometry in case that a specimen is just placed at the focalplane (F=0), and the electron beam is emitted in parallel to the opticalaxis of the system (α=0). In the figure the electron beam is emitted tothe specimen (shown as an arrow) in a direction from the top toward thebottom of the drawing. Part of electron beam will be diffracted withinthe specimen. For instance in case of a crystalline specimen theelectron beam will be dispersed to a specific direction which maysatisfy the Bragg's rule, and the rest thereof will be transmittedthrough the specimen without changing the direction. The objective lensplaced below the specimen may have the characteristics similar to anyordinary optical convex lens, and act to collimate the electron beam.The electrons diffracted to the same direction will be converged to apoint below the lens; the converged electrons forms so-called thediffraction plane (back focal plane). Below the diffraction plane theelectrons diffracted and transmitted at an identical point will beconverged to form the TEM imaging plane. At the TEM focal plane, thesize of the specimen is magnified by M times, depending on theprojection magnification rate M of the objective lens. If F=0 and α=0,then the image of the arrow is correctly focused at the TEM image planeas shown in FIG. 24(a), without displacement from the optical axis(c.f., D=0). If otherwise the specimen is placed at the focus position(F=0) but the electron beam is tilted by an incident angle α, as shownin FIG. 24(b), by means of the wobbler, then the electron beam will besubjected to collimate to another focal position, displaced from theaxis of the objective lens. This may result in a displacement of fieldof view, D=Cs M α³, because of the influence of the spherical aberrationwhich is intrinsic to the convex lens system as similar to the opticallens, where Cs designates to a spherical aberration index which is anintrinsic value of a specific lens. If otherwise the specimen is not atthe focal position and the electron beam is tilted by an incident angleα by means of the wobbler, then the amount of displacement will beworsen as shown in FIG. 24(c). At the amount of defocus F, the positionof specimen will be shifted by an amount a F to the direction normal tothe optical axis the image at the TEM imaging plane will be magnified bythe magnification rate M of the lens to result in a parallax M α F. Thusthe total amount of parallax together with the displacement due to thespherical aberration will become an amount indicated by D=M α (F+Cs α²).It can be clearly seen from this equation that, the parallax D will bezero if α=0, irrelevant whether the specimen is place at the focalposition or not. By using the wobbler in such a manner as describedabove when photographing two pairs of images which have different α eachother, the amount of defocus F may be specifically identified based onthe amount of displacement D in the paired images. The parallax due tothe aberration (Cs M α³) is fewer than the parallax due to the defocus(M α F) in the order of one figure or less. Therefore by minimizing theparallax a focus compensation at higher precision may be achieved. Thefocusing using the wobbler may be considered to be completed if theparallax becomes less than Cs M α³.

[0105] The displacement D in the paired images may be given by using thedisplacement analysis using phase variance of Fourier transform images.

[0106]FIG. 5 shows a schematic diagram describing the displacementanalysis using phase variance of Fourier transform images. Assuming thata pair of images S1 (n, m)=S2 (n+dx, m+dy) has the displacement D=(dx,dy), and the two dimensional discrete Fourier transform of S1 (n, m),and S2 (n, m) is S1′ (k, l), and S2′ (k, l). In accordance with anequation of the Fourier transform, F {S (n+dx, m+dy)}=F {S (n, m) } exp(idxk+idyl), the pair of images may be expressed as S1′ (k, l)=S2′ (k,l) exp (idxk+idyl). The displacement D of S1′ (k, l), and S2′ (k, l) maybe expressed by a phase variance exp (idxk+idyl)=P′ (k, l). Since P′ (k,l) is a wave with the cycle (dx, dy), then in an image P (n, m) which issubjected to invert Fourier transform of a phase variant image P′ (k,l), a δ peak will be appeared at the location (dx, dy). Since it can beassumed that in the image P (n, m) only the δ peak may be present, thecomputation of the center of gravity of the intensity of δ peaks allowsthe correct determination of δ peak even if a fraction is present in theposition of δ peaks.

[0107] When the peak intensity δ is computed after normalizing theintensity of the entire image P (n, m), the intensity will be weaken ifthe unmatched area in the pair of images is larger, in other words, ifthe noise increases. By expressing the peak intensity as the correlationvalue the match between images in the pair may be evaluated.

[0108] By using the apparatus as shown in FIG. 25, focusing will becarried out in accordance with the flow chart shown in FIG. 9. At thebeginning, a field of view on which focus will be analyzed will beselected by the fine-tuning mechanism of the specimen provided by thespecimen stage 18. The selection of a field of view includes also thesetting of magnification rate of the observation and of objectiveaperture. Then the display shown in FIG. 2 will be used to set theoptimal focus, lower correlation threshold, tilt angle α, and repetitionof compensation. Once the parameters are set, a pair of images may betaken by using the electron detector 17. From a pair of images captured,an analyzing image P (n, m) will be derived to identify the peakcorresponding to the displacement. Thus derived displacement D will befurther sent to the computer with control software and image processingsoftware 19. The computer 19 will compute the focus F by means of therelation D=M α (F+Cs α²), and the current of the objective lens I_(obj)to be adjusted corresponding to the focus F, then send this value to theobjective lens control circuit 14′ to carry out the adjustment of thecurrent of the objective lens.

[0109] In this embodiment, exemplary inspections of virus orsemiconductor memory using an electron microscope implementing thefocusing compensator as have been described above will be describedbelow. Since an electron microscope has a capability of resolution inthe level of atoms, may obtain a range of contrasts in accordance withthe structure of the specimen, a variety of observations is performed inbiological as well as non biological fields. In case of viralinspection, the viruses, such as AIDS and Influenza, which are too smallto be identified by an optical microscope, should be identified inshorter times, to determine whether viruses is present or absent, and todiagnose whether the infection is present or absent for a number ofpatients. In such a situation, heretofore, the operator of electronmicroscopy inserts the specimen by hand to the electron microscopeoperating in manual mode to evaluate by naked eyes. The inspection ofsemiconductor memory is another example. A specimen, which was picked upin an appropriate manner and processed to the shape suitable forobservation will be set to an electron microscope and observed. Sincethe density of integration in recent years is increasingly becominghigher than ever and the number of field of view to be observed isincreasing more and more, it is nearly impossible for an inspector tomanually find every defect. In addition, since most of specimens are notmade flat, and are not always placed in a plane perpendicular to theelectron beam, the focusing point will be gradually shifted when thefield of view is continuously transferred one after another, so that thefocusing should be done each time. Accordingly, the throughput of theobservation by means of automatic control of the inspection process isbeing a matter of utmost concern. An example of viral inspection inaccordance with a preferred embodiment of the present invention will bedescribed below in greater details, with reference to FIG. 20, using thefunctionality of automatic focus compensation in accordance with thepresent application. Similarly to the first embodiment above, thedisplacement D will be calculated from a pair of taken images to derivethe amount of defocus F and the current of the objective lens I_(obj)corresponding thereto. Based on these parameters the objective lens willbe immediately readjusted. Thereafter another image will be taken.Otherwise the image for inspection will be taken after repeating thefine adjustment of specimen and the focusing for several times until thetarget field of view will be seen on the image. The comparison with theimage registered for the reference with respect to the viruses to beextracted will be performed thereafter.

[0110] Also in this preferred embodiment, similar to the analysis of thedisplacement D, the consistency of the shapes will be evaluated by usingthe displacement analysis based on the phase variance of two Fouriertransform images to derive the correlation value to determine that thevirus is found therein when the correlation value is less than thepredetermined lower correlation threshold (limit). In this case eitherthe x and y coordinates of the specimen stage 18 on which the viruseshave been found or the specimen number will be stored. If no virus isfound in the field of view then the field of view will be shifted to thenext. To do this each fine tuning mechanism for x-, y-, z-axisrespectively attached to the specimen stage 18 may be used to move it tochange the field of view, or alternatively the deflective coil forcondenser system 16 may be used to transfer the position of electronbeam. Alternatively the fine adjustment mechanism may be provided at theattachment of the electron detector 17 to the electron microscope tomove the electron detector 17 itself. As can be recognized by thoseskilled in the art, the compensation for the transition and drift of theposition of specimen evidently means displacing the position of theelectron beam detector relative to the irradiation point of the electronbeam transmit through the specimen, therefore the most suitable solutionmay be chosen in accordance with the context. There are also a pluralityof solutions for the focusing compensation. In the preferred embodimentas have been described just above, the compensation for focusing may becarried out by adjusting the current of the objective lens to change thefocal distance, however, the compensation alternatively may be performedby detecting the amount of displacement D to finely adjust accordinglythe position of specimen by means of the specimen stage 18 such as inthe direction of incident axis of electron beam in case that thespecimen has been located at the focal position. This alternativesolution is just like that as shown in the flow in FIG. 20 the specimenstage is transferred into the direction of z axis after calculating theamount of defocus F. In case of a drifted specimen either the specimenstage 18 may be moved, or the mounted position of the electron detector17 may be fine-tuned in correspondence with the amount of displacement Din the plane perpendicular to the incident direction of the electronbeam.

[0111] Next, referring to FIG. 21 an example carrying out the preferredembodiment in accordance with the present invention will be describedbelow in greater details. An image (plan view) of an exemplary memorycell, observed from above thereof, transmit from the electron detector17 is as shown in the figure. In most cases the semiconductor chip isconsisted of a number of regular iterative arrays of a given pattern ofshape as shown. A contrast anomaly caused by for example a defect orcontamination debris may be included in part thereof. In FIG. 21, thereare shown defects like line segments and a round contaminant. At firstthe focusing may be adjusted as have been described above, and then thefield will be compared with a registered pattern. An example ofcomparison scheme will be described with reference to FIG. 22. By makinguse of regular pattern in the arrays to be inspected, the field of viewwill be clipped to the size of an elementary pattern. In this situationthe same size of image will be suitable for the registered image to becompared with. Similar to the viral inspection as above, the consistencyof the pattern will be evaluated, and if the correlation value fallsbelow a predetermined lower threshold the corresponding address ofmemory will be recorded. Thereafter the position of clipped field ofview will be shifted to the next to iteratively evaluate the consistencyof the pattern. Once the inspection of the entire image captured hasbeen completed, the field of view will be changed by means of forexample the specimen stage 18 or the deflective coil for condensersystem 16 and the focus will be readjusted to resume the inspection. Inthe foregoing description there are a plurality of patterns of memorycells in the image captured by the electron beam detector at thebeginning, however, it may be possible that the defects to be detectedmay be much smaller, or the contrast of the image may be much lower. Insuch a case it may be required to increase the magnification rate to alevel sufficient to the observation. To do this an image of just onememory cell will be taken with a higher magnification rate to compareone by one with the registered image without clipping. In FIG. 22 thefield of view moves from left hand side to the right hand side on thedrawing sheet. However, any other sequences are equally allowed, andsome examples are shown in FIG. 23.

[0112] The sequence should be chosen in accordance with the performanceof the fine adjuster of the specimen stage and the precision ofdeflection of the deflective coil used.

[0113] In this specification some embodiments by means of a transmissionelectron microscopy (TEM) have been described by way of examples,however, the technology disclosed in the present invention may beequally applied to any other type of inspection apparatus for viewingimages by using charged particle beams such as electrons and ions,including electron microscopes, such as scanning electron microscopes(SEM), scanning transmission electron microscopes (STEM), scanning ionmicroscopes (SIM).

[0114] [Forth Embodiment]

[0115] In an apparatus for observing or inspecting a specimen bycontinuously moving the specimen stage, the transfer of specimen stageat a predetermined constant speed may become difficult if the transferspeed is faster than 5 m/sec due to the error caused by the vibration,inconstant speed, and the precision of transfer rails. This problem canbe solved by the application of the present invention, by using a firstcharged particle beam as the probe to probe the specimen to detect asecond charged particle beam emitted from the specimen to compute theerror of the current position of the specimen stage with the targetposition thereof by using the phase limitation from a plurality ofimages thus obtained to feed back the result to the specimen stage or tothe deflector which deflects the probe before the next image of thespecimen to be inspected in next turn will be captured. This allowsonset of erroneous judgments in the inspection of continuously movingspecimen to be decreased.

[0116] More specifically, The probe, first charged particle beam will becollimated to scan a predetermined area on the specimen through thedeflector and objective lens, then the second charged particle beamemitted from the specimen will be detected by the detector, and thedetected signals will be converted from analog to digital domain tostore in a storing means. The starting point of recording will be heldat a given constant position for every sessions and the scanning will bestarted over again at timing management signals or signal from thespecimen stage or the marking on the specimen. At a predetermined periodof time after the capture of first image, a second image will becaptured. These first and second images will be subjected to the Fouriertransform to determine the phase variance therebetween and will besubjected then to the invert Fourier transform to determine thedisplacement from the origin based on the distance of address in thestorage means to feed the result back to the controller of the specimenstage or to the deflector. The error found in comparison of the firstimage with the second image, due to the malfunction or incorrectoperation during transfer of the specimen stage will be decreased.

[0117] [Fifth Embodiment]

[0118] In the first embodiment described above, an example using a CCDcamera as the imaging device has been disclosed. The fundamentalconfiguration of the CCD camera used is comprised of a scintillator 71,a photo coupler 72, and a CCD camera 73, as have been described abovewith reference to FIG. 6. The images formed on the scintillator 71 willbe focused on the CCD camera 73 at a constant magnification rate ofprojection. In this embodiment, an example using a zoom lens for thephoto coupler 72 to variably set the magnification rate of projection incompliance with the analytic result of the displacement will bedescribed below, with reference to FIG. 27. At the bottom part of theelectron microscope is mounted a electron detector 17 in a mannersimilar to FIG. 19. The detailed configuration is shown in the figure.The electron image made of electron beam will be converted to opticalimage by the scintillator 101 placed in a vacuum. The scintillator 101is adhered on a glass substrate 103, is polished to the most suitablethickness, for example approximately 50 to 120 microns in combinationwith the accelerated electron beam at 100 kV to 400 kV. The opticalimage formed by the scintillator 101 will be focused on the imagingdevice 106 through the optical lens 105. The optical lens 105 andimaging device 106, which are devices of precision structure, arepreferably placed and operated in the atmosphere. In other words onlythe scintillator 101 is installed in the vacuum by using a vacuum seal102, the optical image will be picked up to the atmosphere through theglass substrate 103 which separates the vacuum and the atmosphere. Forthe imaging device 106 a vast majority of two dimensional detectorsincluding not only the CCD device but also any imaging devices such ascamera tubes. The procedure used for determining the amount of defocus Fcorresponding to the displacement D between the first and second imagesis identical to the first embodiment described above In general, thesmaller the amount of defocus F is, the smaller the displacement Dbetween two images. Thus in order to find the amount of displacement Dat a higher precision, an effective way is to increase the magnificationrate of imaging when the displacement D decreased to less than a givenlimit to enlarge the displacement. To increase the magnification rate ofimaging, it will be sufficient to increase the magnification rate of theelectron microscope. However, there may often be arisen undesirableeffects, such as the change of the field of view, change in the imagecontrast resulting from the change of conditions of electron beamoptics. Therefore the inventor have devised a method for changing themagnification rate of imaging without touching the electron microscopy,by changing the magnification of the zoom of the optical lens 105. Anyzoom lens 105 commercially available in the market equipped withmotor-driven zooming mechanism may be used for the optical lens 105. Asshown in the box at the right hand bottom corner of the drawings, themagnification of zooming of the optical lens 105 may be increased to forexample 1.5 fold when the peak indicating the displacement D isapproaching to the origin. If two images at this condition are takenagain, a displacement D′ will be magnified to 1.5 fold accordingly. Ascan be seen from the foregoing description the compensation of focusingas well as drifting may be enabled at higher precision by feeding theresult of analytic images back to the election detector 17.

[0119] [Sixth Embodiment]

[0120] In the first preferred embodiment above a focus compensationusing the parallax has been descried. A stigmatizer, compensator forastigmatic aberration using the parallax may also be achieved. Anastigmatism is a phenomenon that, as shown in FIG. 28, the focus isdistributed to an oval around the optical axis because theelectromagnetic field generated by the objective lens 14 has an ovaldistribution around the optical axis (z-axis). In other words the focushas a distribution described by F (β)=F+A cos² (β−βA), depending on theazimuth β. In this equation F designates to the mean of F (β), referredto as the amount of defocus in general the A designates to the amount ofastigmatism, and β A to the astigmatic orientation.

[0121] The apparatus and the process flow depicted in FIG. 29 may beused to find the focus distribution around the optical axis by the focusanalysis using the parallax to analyze the amount and orientation ofastigmatism to feed the result back to the stigmatizer 141. A first TEMimage will be taken by irradiating the specimen with the incidentelectron beam from a first direction approximately in parallel to theoptical axis, the z-axis, and a second TEM image will be taken byirradiating the specimen with the incident electron beam from a seconddirection tilted by an angle α from the z-axis. The azimuth of thesecond direction with respect to the x-axis will be β₂. The displacementD (β₂) between the first TEM image and the second TEM image will beanalyzed by the displacement analysis processor using phase variance ofFourier transform images 20, which will in turn send thus founddisplacement D (β₂) to the computer 19, which further will computes theamount of defocus F (β₂) at the azimuth β₂. Thereafter, a third TEMimage will be taken by irradiating the specimen with the electron beamfrom a third direction where the direction is tilted by angle α from thez-axis, and the azimuth to the x-axis becomes β₃, in order to analyzethe displacement D (β₃) between the first TEM image and the third TEMimage to determine the amount of defocus F (β₃) at the azimuth β₃ to thex-axis. Then the same procedure of analysis will be applied to aplurality of azimuths β_(n) to determine the distribution of azimuth atthe focal point.

[0122] Any one of fitting methods including such as least-square methodwill be used to identify the amount of defocus, astigmatism, and theastigmatic orientation from the focal point F (β_(n)) at eachorientation. With respect to the astigmatic orientation there may be acase in which the azimuth of incident electron beam may not be inparallel to the direction of displacement vector, by the influence ofimage rotation being generated within the electron lenses. Since thedifference between the orientation of incident electron beam and thedirection of displacement vector may be determined once conditions oflens such as the magnification rate has been decided, the difference ateach of lens conditions may be stored in the computer to correct theastigmatic orientation based on the stored difference.

[0123] Based on the result of the astigmatism analysis, the currentvalues I sx and I sy of the stigmatizer 141, required in order for theamount of astigmatism A to become zero, will be computed so as to adjustthe stigmatizer 141 through the stigmatizer control circuit 141′.

[0124] The astigmatic analysis needs a precision by two digits or moresuperior to the precision of defocus analysis. In a conventional focusanalysis system using the cross-correlation for the analysis ofdisplacement it will be very difficult to satisfy a precision requiredto carry out the stigmation. The analysis of astigmatism is an analysisof focal distribution, so that a large number of times of displacementanalysis will be required. The system disclosed herein, equipped withthe displacement analysis processor using phase variance of Fouriertransform images 20, based on a digital signal processor (DSP), mayperform one focusing analysis within a second, allowing one analysis ofastigmatism to be completed within a few seconds.

[0125] The performance of apparatus compensating for an electronmicroscope based on the displacement between the images taken by theelectron microscope such as the focusing analysis using the parallax islargely depending on the displacement analysis. In the analysis methodof displacement used in the conventional compensator systems theprecision of analysis in theory cannot be smaller than the size of apixel of the electron detector 17. However, in accordance with thepresent invention, a precision of analysis smaller than the size of apixel may be obtained. The apparatus in accordance with the presentinvention is capable to adjust the focusing at a precision as fine as askillful operator. Although the time required for analysis and the costof hardware will be increased when improving the performance in anattempt to improve the precision of focal analysis, such as subdividingthe image to be inspected into still smaller pieces, the presentinvention, which may improve the precision of the analysis ofdisplacement by altering the analysis method of displacement, allows theprecision of focusing analysis to be improved without additional time ofanalysis and cost of hardware.

[0126] Furthermore, in accordance with the present invention, theconsistency between paired images is indicated as a correlation. Theoperator of the electron microscope may check the reliability of theanalytic result output. Incorrect operation may be prevented by settinga lower threshold of correlation values so as not to limit theadjustment of the lens system when a calculated correlation value isless than the lower threshold. In an automatic inspection apparatus, theoperator may check to see later whether or not the automaticcompensation has been performed correctly, by storing the correlationvalues in the focal analysis and the results of focal analysis, allowingunmanned operation to be performed.

[0127] The analysis of displacement in accordance with the presentinvention is a method making use of the phase component of images, whichis almost immune to the variance of background, and is operable if thereis a sufficient common area of paired images, even when some extent ofshadow of aperture covers the images. The system in accordance with thepresent invention is still operable when the TEM is not sufficientlyconfigured. In brief, an operator unfamiliar with the operation of TEMmay use the system.

[0128] The foregoing description of some preferred embodiments of theinvention has been presented for purposes of illustration anddescription. It is not intended to be exhaustive or to limit theinvention to the precise forms disclosed, and modifications andvariations are possible in light of the above teachings or may beacquired from practice of the invention. The embodiments are chosen anddescribed in order to explain the principles of the invention and itspractical application to enable one skilled in the art to utilize theinvention in various embodiments and with various modifications as aresuited to the particular use contemplated it is intended that the scopeof the invention be defined by the claims appended hereto, and theirequivalents.

What is claimed is:
 1. An electron beam apparatus, comprising: a sourceof charged particle beam; a specimen stage for holding a specimen; aspecimen stage controller for moving the specimen stage; a deflectorcoil for deflecting an incident position of a first charged particlebeam into the specimen; a detector for detecting a second chargedparticle beam from the specimen and generating an image signal; acomputer for obtaining a plurality of images from the image signal tocalculate a transfer speed of the specimen; and a deflector coilcontroller for controlling the operation of the deflector coil so as tocancel out the influence of transfer of the specimen stage.
 2. Anelectron beam apparatus according to claim 1, wherein: the specimenstage controller moves the specimen stage at a certain velocity; and thedeflector coil controller controls the deflector coil to shift thesecond charged particle beam at a substantially same velocity as thespecimen stage.
 3. An electron beam apparatus according to claim 1,wherein: the specimen stage controller stops the specimen stage; and thedeflector coil controller controls the deflector coil to shift thesecond charged particle beam so as to cancel out a specimen drift.
 4. Amethod of operating an electron microscope, comprising the steps of:placing a specimen on a specimen stage; continuously moving the specimenstage; irradiating the specimen with a first electron beam; taking animage by a second electron beam from the specimen; capturing a pluralityof taken images for storage; performing an image processing on saidplurality of taken images to determine a consistency between theplurality of taken images; determining an amount of displacement betweenthe plurality of taken images; and controlling a deflector coil todeflect the irradiation of the first electron beam to the specimen so asto cancel out the transfer of the specimen stage based on the determinedconsistency and displacement.